The goal of this project is to elucidate the mechanism of electron transfer in the Rieske iron-sulfur protein of the bc1 complex. The bc1 complex links the energy of electron transfer to proton transport across the inner mitochondrial membrane in a process called the Q-cycle. The Q-cycle has been documented by extensive experimental evidence, however it is unknown how the bc1 complex performs the electron transfer and protonmotive activities at the molecular level. An understanding of how the bc1 complex performs this energy transduction is essential to understanding cellular energetics. The Q-cycle mechanism places constraints on the midpoint potential of the iron-sulfur protein. If the potential becomes too positive, the iron-sulfur protein will still oxidize ubiquinol, but be unable to reduce cytochrome c1. If the potential becomes too much less positive, the iron-sulfur protein will be unable to oxidize ubiquinol, and thus prevent the oxidant-induced reduction of cytochrome b. By altering the midpoint potential of the iron-sulfur protein insight into the structural properties of the iron-sulfur protein, and it s role in the Q-cycle mechanism will be obtained. Site-directed mutagenesis will be utilized to change residues in the vicinity of the iron-sulfur cluster in order to change it s midpoint potential. These mutant proteins will be expressed in a mutant S. cerevisiae strain in which the endogenous iron-sulfur protein gene has been deleted. The transformants will be tested for growth on a non- fermentable carbon source, and ubiquinol-cytochrome C oxidoreductase activity will be measured. The presence of the mature protein in mitochondrial membranes will be determined by western analysis, and the presence of the [2Fe-2S] cluster will be determined by circular dichroism (CD). If the cluster is present, CD will be used to measure the midpoint potential, and the pre-steady state kinetics of electron transfer from ubiquinol to cytochrome b and c1 will be measured. Another aim is to elucidate the function of the disulfide in the cluster-binding fold. Site-directed mutagenesis will be used to change these cysteine residues, and the mutant proteins will be analyzed as previous described.